PROJECT SUMMARY Cryptosporidiosis is amongst the most important causes of life-threatening diarrhea in children globally, causes incurable diarrhea in AIDS and transplant patients, and is the most common cause of waterborne diarrheal outbreaks in the United States. Almost all human cases of cryptosporidiosis are due to infection of the small intestinal epithelium with one of two species of Cryptosporidium parasites, C. parvum and C. hominis. Nitazoxanide, the only approved drug, is efficacious in otherwise healthy adults, but unfortunately, has limited efficacy (~56%) in children and is equivalent to a placebo in AIDS patients. The goal of this project is to address the need for new anti-Cryptosporidium drugs via an exciting collaboration between the German pharmaceutical company EMD-Serono, an academic Cryptosporidium biologist, an academic medicinal chemist with extensive prior industry experience, and an academic biologist (also with extensive industry experience) whose laboratory specializes in in vitro and in vivo drug pharmacology. The developmental cascade to accomplish this is guided by an ideal target product profile for a drug effective in all affected patient populations; the methods bring together novel in vitro assays and a highly immunocompromised mouse model of cryptosporidiosis with well-established pharmacology and medicinal chemistry approaches. The R21 phase will deliver two lead compounds with anti- Cryptosporidium efficacy in a chronic mouse model of infection, along with knowledge of key compound characteristics and potential safety concerns. The open access EMD-Serono ?mini-library? has already been screened and three promising Cryptosporidium growth inhibitors were identified that are suitable for follow-up. A second EMD-Serono mini-library will be screened to identify additional potential starting points. Validated Cryptosporidium growth inhibitors will then be prioritized using 1) assays to assess if compounds are cidal or static, selective, and active against C. hominis, and 2) existing EMD-Serono pharmacokinetic and safety data and available EMD-Serono analogs for structure-activity relationship (SAR) studies. After prioritization and testing available analogs in vitro, mouse pharmacokinetic studies and efficacy studies will be performed. If the progression milestones are met, the R33 phase will result in optimization of one lead chemical series for potency and safety. The other series will be held in backup. For this, cyclic rounds of chemical synthesis will be combined with in vitro Cryptosporidium assays, in vitro ADME studies, mouse PK studies, and the chronic mouse model of cryptosporidiosis. Success would yield an optimized clinical candidate that is ready to be advanced to testing in large animal diarrhea models and regulatory toxicology studies. Given the dire need for new cryptosporidiosis drugs, the public health impact of success could be extremely significant.